SPOT Peptide Array Research Articles

Discovery of NSD2 non-histone substrates and design of a super-substrate

The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.

Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues.

The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3 -R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.

Identification of GDF15 peptide fragments inhibiting GFRAL receptor signaling

Growth differentiation factor 15 (GDF15) is believed to be a major causative factor for cancer-induced cachexia. Recent elucidation of the central circuits involved in GDF15 function and its signaling through the glial cell-derived neurotrophic factor family receptor α-like (GFRAL) has prompted the interest of targeting the GDF15-GFRAL signaling for energy homeostasis and body weight regulation. Here, we applied advanced peptide technologies to identify GDF15 peptide fragments inhibiting GFRAL signaling. SPOT peptide arrays revealed binding of GDF15 C-terminal peptide fragments to the extracellular domain of GFRAL. Parallel solid-phase peptide synthesis allowed for generation of complementary GDF15 peptide libraries and their subsequent functional evaluation in cells expressing the GFRAL/RET receptor complex. We identified a series of C-terminal fragments of GDF15 inhibiting GFRAL activity in the micromolar range. These novel GFRAL peptide inhibitors could serve as valuable tools for further development of peptide therapeutics towards the treatment of cachexia and other wasting disorders.

Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

Identification of nonhistone substrates of the lysine methyltransferase PRDM9

Lysine methylation is a dynamic, post-translational mark that regulates the function of histone and non-histone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate non-histone proteins. In this work, we investigate the substrate selectivity of the lysine methyltransferase PRDM9 to identify both potential histone and non-histone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on non-histone proteins. Using lysine-oriented peptide (K-OPL) libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative non-histone substrates, which were tested by peptide spot array, and a subset were further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the non-histone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.

The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site.

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.

Identification of Eral1 Recognition Sequences for Unfolding and Degradation by Mitochondrial ClpXP

Eral1 is a GTPase and ribosomal assembly factor of the mitochondrial small subunit (SSU). Eral1 binds 12SrRNA at a critical juncture, and its association and dissociation are necessary for mitoribosome maturation. Previous work in the literature indicated the proteolytic complex mitochondrial ClpXP (mtClpXP) as a regulator of dissociation: mtClpXP physically associates with Eral1 in vivo and knockout of ClpP results in Eral1 stabilization, accumulation of Eral1‐bound small subunit precursors, and reduced levels of mature mitoribosomes. In order for mtClpXP to both permit Eral1 association and enact its dissociation from the assembling small subunit, it must either recognize a specific Eral1 conformation that indicates completion of its assembly factor function or precisely tune Eral1 levels to achieve a metastable state. We aim to describe the recognition strategy that mtClpXP utilizes to unfold and degrade Eral1 and enable ribosome assembly. We have demonstrated that mtClpXP directly unfolds and degrades Eral1 in vitro. Evolutionary coupling analysis between human Eral1 and mtClpX has pointed to select residues that may mediate the interaction between these proteins. We have shown that mutation of at least one such Eral1 residue blocks its unfolding and degradation. We are utilizing a peptide SPOT array to characterize additional Eral1 motifs that assist in recognition and unfolding by mtClpX and hypothesize that the exposure of these sequences aligns with the Eral1 state that is recognized by mtClpX.

Specificity Analysis of Protein Methyltransferases and Discovery of Novel Substrates Using SPOT Peptide Arrays.

Posttranslational methylation of amino acid side chains in proteins mainly occurs on lysine, arginine, glutamine, and histidine residues. It is introduced by different protein methyltransferases (PMTs) and regulates many aspects of protein function including stability, activity, localization, and protein/protein interactions. Although the biological effects of PMTs are mediated by their methylation substrates, the full substrate spectrum of most PMTs is not known. For many PMTs, their activity on a particular potential substrate depends, among other factors, on the peptide sequence containing the target residue for methylation. In this protocol, we describe the application of SPOT peptide arrays to investigate the substrate specificity of PMTs and identify novel substrates. Methylation of SPOT peptide arrays makes it possible to study the methylation of many different peptides in one experiment at reasonable costs and thereby provides detailed information about the specificity of the PMT under investigation. In these experiments, a known substrate sequence is used as template to design a SPOT peptide array containing peptides with single amino acid exchanges at all positions of the sequence. Methylation of the array with the PMT provides detailed preferences for each amino acid at each position in the substratesequence, yielding a substrate sequencespecificity profile. This information can then be used to identify novel potential PMT substrates by in silico data basesearches. Methylation of novel substrate candidates can be validated in SPOT arrays at peptide level, followed by validation at protein level in vitro and in cells.

Assessing biofilm inhibition and immunomodulatory activity of small amounts of synthetic host defense peptides synthesized using SPOT-array technology.

Peptides are promising drug candidates because of their diversity, biocompatibility and spectrum of activities. Here, we describe a protocol for high-throughput screening of SPOT-peptide arrays to assess the antibiofilm, antimicrobial and immunomodulatory activities of synthetic peptides. It is a Protocol Extension of our previous Nature Protocols article, which describes the synthesis of SPOT-peptide arrays and assays for screening antimicrobial activity. This latest protocol allows the simultaneous assessment of hundreds of synthetic host defense peptides to define their overall activity profiles and identify candidate sequences that are suitable for further characterization and development as anti-infectives. When coupled with the SPOT-array technology for peptide synthesis, the described procedures are rapid, inexpensive and straightforward for peptide library screening. The protocols can be implemented in most microbiology or immunology research laboratories without the need for specialists. The time to complete each step ranges between 1 and 4 h with overnight pauses, and datasets related to the antibiofilm and immunomodulatory activities of a large set of peptide sequences can be generated in a few days.

The vimentin rod domain blocks P-selectin-P-selectin glycoprotein ligand 1 interactions to attenuate leukocyte adhesion to inflamed endothelium.

Acute inflammation begins with leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin on inflamed endothelium and platelets. In pathologic conditions, this process may contribute to secondary organ damage, like sepsis-induced liver injury. Therefore, developing novel therapies to attenuate inflammation may be beneficial. We previously reported that recombinant human vimentin (rhVim) binds P-selectin to block leukocyte adhesion to endothelium and platelets. In this study, we used SPOT-peptide arrays to identify the rod domain as the active region within rhVim that interacts with P-selectin. Indeed, recombinant human rod domain of vimentin (rhRod) binds to P-selectin with high affinity, with in silico modeling suggesting that rhRod binds P-selectin at or near the PSGL-1 binding site. Using bio-layer interferometry, rhRod decreases PSGL-1 binding to immobilized P-selectin, corroborating the in silico data. Under parallel-plate flow, rhRod blocks leukocyte adhesion to fibrin(ogen)-captured platelets, P-selectin/Fc-coated channels, and IL-1β/IL-4-co-stimulated human umbilical vein endothelial cells. Finally, using intravital microscopy in endotoxemic C57Bl/6 mice, rhRod co-localizes with P-selectin in the hepatic sinusoids and decreases neutrophil adhesion to hepatic sinusoids. These data suggest a potential role for rhRod in attenuating inflammation through directly blocking P-selectin-PSGL-1 interactions.

Systematic Screening and Deep Analysis of CoPt Binding Peptides Leads to Enhanced CoPt Nanoparticles Using Designed Peptides.

Using protein and peptide additives to direct the crystallization of inorganic materials is a very attractive and environmentally friendly strategy to access complex and sometimes inaccessible mineral phases. CoPt is a very desirable high-magnetoanisotropic material in its L10 phase, but this is acquired by annealing at high temperatures which is incompatible with delicate nanomaterial assembly. Previous studies identified one peptide with high affinity to CoPt and four peptides with high affinity to FePt L10 phase nanoparticles (NPs) through phage display biopanning selection. While synthesis mediated by these peptides offered a small degree of L10 character to the NPs, they do not have the magnetoanistropy required for applications. In this study, we improve the activity of peptide directed crystallization by designing second generation peptides. We use the five literature sequences (LS) to probe the binding affinity deeper through dissection (alanine scanning), reduction (truncations), and substitution of the LS to find key amino acids and motifs. This is performed using a SPOT peptide array, importantly probing interactions at three stages of NP formation: with precursor, during synthesis, and with NPs. We found four universal features: 1) the importance of basic residues, particularly lysine flanking both ends of the sequence; 2) the importance of methionine; 3) shorter sequences show higher affinity than longer ones; and 4) acidic residues have a negative impact on binding with aspartic acid less favorable than glutamic acid. However, an acidic amino acid benefits, presumably to balance charge. The short motif KSLS had high affinity in all assays. Three sequences were selected from the screening, and three sequences were designed from the rules above. These were used to mediate a green synthesis of CoPt nanoparticles. The screened peptides mediated the formation of NPs with improved coercivity (90-110 Oe) compared to the LS (30-80 Oe), while the designed peptides facilitated formation of CoPt NPs with the highest coercivity (109 to 132 Oe), representing a massive improvement on L10 character. This result along with deeper insight this methodology brings offers vast potential for the future.

Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions.

Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the "holdup" principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities.

Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1.

The cAMP-dependent protein kinase A (PKA) regulates various cellular functions in health and disease. In endothelial cells PKA activity promotes vessel maturation and limits tip cell formation. Here, we used a chemical genetic screen to identify endothelial-specific direct substrates of PKA in human umbilical vein endothelial cells (HUVEC) that may mediate these effects. Amongst several candidates, we identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1α at Ser268 and ATG16L1β at Ser269, driving phosphorylation-dependent degradation of ATG16L1 protein. Reducing PKA activity increased ATG16L1 protein levels and endothelial autophagy. Mouse in vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. Together these results indicate that endothelial PKA activity mediates a critical switch from active sprouting to quiescence in part through phosphorylation of ATG16L1, which in turn reduces endothelial autophagy.

In Silico Engineering of Synthetic Binding Proteins from Random Amino Acid Sequences

SummarySynthetic proteins with high affinity and selectivity for a protein target can be used as research tools, biomarkers, and pharmacological agents, but few methods exist to design such proteins de novo. To this end, the In-Silico Protein Synthesizer (InSiPS) was developed to design synthetic binding proteins (SBPs) that bind pre-determined targets while minimizing off-target interactions. InSiPS is a genetic algorithm that refines a pool of random sequences over hundreds of generations of mutation and selection to produce SBPs with pre-specified binding characteristics. As a proof of concept, we design SBPs against three yeast proteins and demonstrate binding and functional inhibition of two of three targets in vivo. Peptide SPOT arrays confirm binding sites, and a permutation array demonstrates target specificity. Our foundational approach will support the field of de novo design of small binding polypeptide motifs and has robust applicability while offering potential advantages over the limited number of techniques currently available.

Biochemical analysis and identification of linear B-cell epitopes from recombinant Sm21.7 antigen from Schistosoma mansoni

Schistosoma mansoni tegument is a dynamic host-interactive layer that is an essential source of parasite antigens and a relevant field for schistosome vaccine research. Sm21.7 is a cytoskeleton antigen found in S. mansoni tegument that engenders protection in experimental challenge infection. Because of its crucial role in the parasite tegument and its promising protective capability, Sm21.7 is an exciting target for the development of therapeutic strategies. The present study describes Sm21.7 structural and biophysical features using circular dichroism spectroscopy and identifies linear B-cell epitopes of Sm21.7 using in-silico methods and immunoassay. The Sm21.7 gene was cloned into the pETDEST42 vector, and the recombinant protein was overexpressed in Escherichia coli DE3. The soluble protein was purified by affinity chromatography followed by ion-exchange chromatography. Purified recombinant Sm21.7 was analyzed by circular dichroism spectroscopy which demonstrated that the rSm21.7 structure was comprised of approximately 38% α-helices and its conformation remains stable at temperatures of up to 60 °C. Prediction of rSm21.7 B-cell epitopes was based on amino acid physicochemical properties. Sixteen peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by spot peptide array using pooled rSm21.7-immunized mice sera or patients’ sera with different clinical forms of S. mansoni infection. Immunoassays revealed that sera from rSm21.7-immunized mice reacted predominantly with peptides located in the dynein-light chain domain (DLC) at the C-terminal region of rSm21.7. Comparative analysis of the antibody response of acute, intestinal and hepatosplenic patients’ sera to the Sm21.7 peptides showed that a differential recognition pattern of Sm21.7-derived peptides by intestinal patients’ sera might contribute to down-regulate the immune response in chronic intestinal patients. Together, the results may help the development of S. mansoni vaccine strategies based on the rSm21.7 antigen.

An optimized method using peptide arrays for the identification of in vitro substrates of lysine methyltransferase enzymes

While a number of post-translational modifications (PTM), such as phosphorylation and ubiquitination, have been extensively studied, lysine methylation is emerging as an important PTM with implications in a growing number of diverse cellular processes. To date, there are approximately 5000 identified methylation sites on non-histone proteins, and as the methyllysine proteome expands it becomes important to identify the lysine methyltransferase enzymes responsible for each methylation event. The use of peptide SPOT methylation assay has proven to be a useful in the identification and validation of novel substrates for lysine methyltransferase enzymes as it uses a weak beta emitter coupled with fluorography to detect methylation events. The method described in this paper provides improvements to the typical protocol for this assay, as a highly sensitive tritium assay can be developed with less radioactivity than previously described. This protocol provides an inexpensive alternative to weak beta signal enhancer sprays and washes for use in lysine methylation peptide SPOT arrays, and a simple open-source method for array quantification.

Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment

The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3′ untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex “carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT),” which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3′-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay.

Application of mixed peptide arrays to study combinatorial readout of chromatin modifications

The N-terminal tails of histone proteins are massively decorated with post-translational modifications (PTMs), which play important roles in the regulation of gene expression. Several highly conserved chromatin interacting proteins can bind to histone modifications in a sequence and modification specific manner employing specific reading domains. These proteins often contain several reading domains, which can cooperate in the readout of different PTMs. To gain a better insight into the combinatorial readout of PTMs, we developed a method to study the binding of double reading domains to mixed peptide arrays containing two different peptides in each spot. For that, differently modified and unmodified peptides were prepared by SPOT synthesis and solubilized. Then, two peptides were mixed and spotted onto a glass slide creating peptide spots presenting two modifications on two different peptides. Different combinations of mixed spots containing modified and unmodified peptides were generated and incubated with recombinant double reading domains to study their synergistic binding. For validation of the method, we used the well-studied BPTF subunit of the NURF chromatin-remodeling complex. BPTF contains a plant homeodomain finger (PHD) and a Bromodomain recognizing H3K4me3 and H4K16ac, respectively. We first confirmed with peptide arrays and Fluorescence Anisotropy (FA) measurements that the BPTF PHD-Bromo (PB) domain interacts specifically with the expected modifications. Using our novel tool, we observed a strong and synergistic binding only to peptide spots containing both modifications, which was lost if one of the domains was inactivated by a mutation. These data indicate that BPTF-PB simultaneously interacts with both target modifications using its PHD and Bromodomain. In agreement with the synergistic peptide interaction on mixed peptide arrays, we also show that chromatin pulldown by BPTF-PB depends on the activity of both reading domains. We conclude that mixed peptide spot arrays are a powerful, cheap and novel method for screening the combinatorial interaction space of multidomain reading proteins. Using this approach hundreds of mixed peptide spots can be prepared and tested for binding in principle allowing for an unbiased medium throughput investigation.

Mapping the recognition domains of pneumococcal fibronectin-binding proteins PavA and PavB demonstrates a common pattern of molecular interactions with fibronectin type III repeats.

Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin-binding proteins (FnBPs) PavA and PavB respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non-human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both β-sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII1 , FnIII5 or FnIII15 bind directly to FnBPs PavA and PavB respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours.

CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell‐Specific Adaptor Protein

The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr)629 and linker of activated T cells (LAT) pTyr171 , pTyr191 and pTyr226 .